TIMP1 as a marker for cholangiocarcinoma

Abstract

The present invention relates to an in vitro method for assessing cholangiocarcinoma in a patient sample, the method comprising: a) determining the level of tissue inhibitor of metalloproteinase 1 (TIMP1) in the patient sample, the patient sample being selected from the group consisting of serum, plasma, and whole blood samples from an individual; b) comparing the level of TIMP1 determined in step (a) with a reference level of TIMP1; and c) assessing cholangiocarcinoma in the patient sample by comparing the level determined in step (a) with the reference level of TIMP1, the level being indicative of cholangiocarcinoma in the patient sample. The present invention further relates to an in vitro method for assessing cholangiocarcinoma comprising TIMP1 and MMP2, the use of TIMP1 and optionally MMP2 in the in vitro assessment of CCA, and a kit for carrying out the method.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to an in vitro method for assessing cholangiocarcinoma in patient samples and a kit for carrying out said method.Furthermore, the present invention relates to the use of TIMP1 as a marker molecule and a marker combination comprising TIMP1 and MMP2, respectively, in the in vitro assessment of cholangiocarcinoma. [Background technology]

[0002] 2. Background of the Invention Cholangiocarcinoma (CCA or CCC) is a highly lethal malignant biliary tumor arising from the epithelium of the bile duct. CCA accounts for approximately 3% of all gastrointestinal tumors and is the second most common primary liver tumor after hepatocellular carcinoma (HCC). Based on its anatomical origin, CCA is classified as intrahepatic (iCCA), perihilar (pCCA), or distal (dCCA) CCA (Rimola J. et al., Hepatology, 2009, 50(3):791-798). The incidence of CCA, especially iCCA, appears to be increasing and is known to be as high as 2.1 per 100,000 person-years in the Western world and highest in northeastern Thailand (>80 per 100,000; Martha M. Kirstein, Arndt Vogel, Epidemiology and Risk Factors of Cholangiocarcinoma, Visc Med 2016;32:395-400).

[0003] CCA has similar risk factors as HCC, including cirrhosis, chronic viral hepatitis, alcohol excess, diabetes and obesity. Further well-established risk factors for CCA are related to chronic biliary inflammation, such as hepatobiliary flukes, primary sclerosing cholangitis (abbreviated as PSC), biliary cysts, hepatolithiasis and toxins (Bridgewater J.et al., J Hepatol.2014,6:1268-893,8-14; Khan SAet al., Consensus document.Gut.2002,51 Suppl 6:VI1-9; Khan SAet al., Gut.2012,61(12):1657-1669; Alvaro D.et al., Dig Liver Dis.2010,42:831-838; Benavides M.et al., Clin Transl Oncol.2015,17:982-987; Benson III ABet al., J Natl Compr Canc Netw.,2009,7:350-391;Cai JQet al.,J Huazhong Univ Sci Technolog Med Sci.,2014,34:469-475;Rerknimitr R.et al.,J Gastroenterol Hepatol.,2013,28:593-607).

[0004] CCA diagnosis is often complex and requires the use of multiple diagnostic modalities to distinguish benign from malignant structures, differentiate CCA from other primary liver tumors, mainly HCC and combined HCC-CCA, and stage and grade the tumor. Differentiation between CCA, especially iCCA, and HCC is essential for surgical planning and prognostic assessment. Imaging methods, such as dynamic computed tomography scans, can differentiate HCC from CCA based on the absence of heterogeneous contrast uptake and contrast efflux in the delayed phase, characteristic of cholangiocarcinoma (Rimola J. et al., Hepatology 2009,50(3):791-798). However, these classical features of intrahepatic cholangiocarcinoma are not universally present in all CCA cases (Iavarone M. et al., J Hepatol., 2013, 58:1188-93; Kim SH et al., J Comput Assist Tomogr., 2012, 36:704-09).

[0005] To date, there is no specific blood test available for the specific diagnosis of CCA. Carbohydrate antigens CA19-9 and CA-125, as well as carcinoembryonic antigen CEA, are the most widely used markers for suspected CCA, but their diagnostic capabilities are very limited. CA19-9 is elevated in pancreatic, colorectal, and gastric cancer, as well as non-malignant conditions such as PSC (primary sclerosing cholangitis) and obstructive jaundice. Furthermore, patients lacking Lewis antigens (10% of the general population) are unable to produce CA19-9 and therefore do not benefit from the test. The sensitivity and specificity of CA19-9 in CCA patients range from 40% to 70% and 50% to 80%, respectively, with a positive predictive value of 16% to 40%. CEA is elevated in 20-30% of CCA patients, whereas CA-125 is elevated in approximately 40%-50%, which is insufficient for an accurate diagnosis (Alsaleh M.et al.,Int J Gen Med.,2018,12:13-23; Khan SAet al.,Gut.,2002,51 Suppl 6:VI1-9; Patel AHet al.,Am J Gastroenterol,2000,95:204-207; Hultcrantz R.et al.,J Hepatol,1999,30:669-673).These limitations are recognized by the major international guidelines, and CEA and CA19-9 measurements are only recommended as supportive tools in the management of cholangiocarcinoma, but not as diagnostic tools (Khan SA et al., Consensus document. Gut., 2002, 51 Suppl 6:VI1-9; Khan SA et al., Gut. 2012, 61(12):1657-1669; Alvaro D. et al., Dig Liver Dis., 2010, 42:831-838; Benavides M. et al., Clin Transl Oncol., 2015, 17:982-987; Benson III AB et al., J Natl Compr Canc Netw., 2009, 7:350-391; Cai JQ et al., J Huazhong Univ Sci Technolog Med Sci., 2014, 34: 469-475; Rerknimitr R. et al., J Gastroenterol Hepatol., 2013, 28: 593-607).

[0006] Therefore, the identification of novel biomarkers for more specific diagnosis and stratification of CCA is recognized as one of the unmet medical needs by the current EASL guidelines for the diagnosis and management of intrahepatic cholangiocarcinoma (Bridgewater J., J Hepatol., 2014, 6:1268-89).

[0007] As discussed above, all current methods for the diagnosis and differential diagnosis of bile duct carcinoma have significant drawbacks.

[0008] Ultrasound: Ultrasound examination (abbreviated as US) is reliable to exclude gallstones, but is operator-dependent and insufficient to investigate a suspected CCA alone. US may miss small tumors and cannot accurately define the extent of the tumor (Khan SA et al.,Gut.,2002,51 Suppl 6:VI 1-9; Khan SA et al.,Gut.2012,61(12):1657-1669). Only the Japanese guidelines and the 2002 edition of the BSG guidelines recommended US as an initial test included in the diagnostic algorithm (Khan SA et al.,Gut.,2002,51 Suppl 6:VI 1-9; Khan SA et al.,Gut.,2012,61(12):1657-1669).

[0009] Contrast-enhanced ultrasound (abbreviated as CEUS) significantly improves diagnostic performance in distinguishing intrahepatic cholangiocarcinoma (iCCA) from HCC compared with baseline ultrasound, but the performance is highly reader-dependent, with AUCs ranging from 0.650 to 0.933, as reported by Chen LD et al., Eur Radiol., 2010, 20(3):743-53. Furthermore, the usefulness of CEUS in diagnosing small ICCs is unknown.

[0010] High-resolution / spiral computed tomography (abbreviated as CT): Contrast-enhanced CT has a higher sensitivity (up to 80%) than US for detecting CCA and provides a good view of intrahepatic mass lesions, dilated intrahepatic ducts, focal lymphadenopathy and extrahepatic metastases. However, unlike HCC (Sun H. and Song T. Drug Discov Ther., 2015, 9: 310-318), radiological criteria of CT or magnetic resonance imaging (abbreviated as MRI) are insensitive to the diagnosis of CCA. Therefore, pathological diagnosis is required for the definitive diagnosis of CCA. In addition, CT / MRI may miss small lesions (Chen LD et al., Eur Radiol., 2010, 20(3): 743-53; Hanninen ELet al., Acta Radiol., 2005, 46: 462-470).

[0011] Serum tumor markers: Carbohydrate antigens CA19-9 and CA-125, and carcinogenic antigen CEA are the most used serum tumor markers. Both of these blood tumor markers have significant overlap with other benign diseases and low sensitivity for early stage disease, limiting their use for diagnosis. The sensitivity and specificity of CA19-9 for iCCA is only 62% and 63%, respectively. The diagnostic performance of CEA is even lower, as it is elevated in only 20-30% of CCA patients, whereas CA-125 is elevated in approximately 40%-50%, which is insufficient for accurate diagnosis (Alsaleh M.et al.,Int J Gen Med.,2018,12:13-23; Khan SAet al.,Gut.,2002,51 Suppl 6:VI1-9; Patel AHet al.,Am J Gastroenterol,2000,95:204-207; Hultcrantz R.et al.,J Hepatol,1999,30:669-673).

[0012] Whole blood, serum or plasma are the most widely used sources of patient samples in clinical routine. Identification of early CCA markers that aid in reliable CCA detection or provide early prognostic information may lead to valuable methods for the diagnosis and management of this disease.

[0013] Therefore, there is an urgent clinical need to improve the in vitro assessment of CCA. Improved early diagnosis of CCA is particularly important because patients diagnosed at an early stage of CCA have a much higher chance of reversibility of bile duct damage compared with patients diagnosed at a more advanced stage of the disease.

[0014] There is a strong need in the art to identify reliable and direct indicators of CCA disease state to reliably distinguish symptoms of CCA from those of the other respiratory diseases mentioned above, and to predict changes in disease severity, disease progression and response to drugs.

[0015] The object of the present invention is to provide a simple and cost-effective procedure for CCA assessment, for example to identify individuals suspected of having CCA. In particular, the object of the present invention is to provide an in vitro method for assessing cholangiocarcinoma in patient samples and a kit for carrying out said method. Furthermore, the object of the present invention relates to the use of TIMP1 as a marker molecule and a marker combination comprising TIMP1 and MMP2, respectively, in the in vitro assessment of cholangiocarcinoma.

[0016] This object or these objects are solved by the subject matter of the independent claims. Further embodiments are subject to the dependent claims. Summary of the Invention

[0017] Summary of the Invention It has now been found that the use of tissue inhibitor of metalloproteinase 1 (TIMP1) can at least partially overcome some of the problems of currently known methods available for the assessment of CCA.

[0018] Surprisingly, it has been found in the present invention that an in vitro method comprising determining the level of TIMP1 in a patient sample allows for the assessment of CCA.In this connection, it has been found that an increase in the level of TIMP1 in such a sample obtained from an individual compared to the reference level of TIMP1 indicates the presence of CCA.

[0019] In a first aspect, the present invention provides an in vitro method for assessing cholangiocarcinoma in a patient sample, comprising: a) determining the level of tissue inhibitor of metalloproteinase 1 (TIMP1) in a patient sample, the patient sample being selected from the group consisting of serum, plasma and whole blood samples from an individual; b) comparing the level of TIMP1 determined in step (a) with a reference level of TIMP1; and c) assessing cholangiocarcinoma in the patient sample by comparing the level determined in step (a) with a reference level of TIMP1, wherein an increased level of TIMP1 compared to the reference level of TIMP1 is indicative of cholangiocarcinoma in the patient sample.

[0020] In a second aspect, the present invention relates to the use of TIMP1 as a marker molecule for the in vitro assessment of cholangiocarcinoma in a serum, plasma or whole blood sample of an individual, where a level of TIMP1 above a reference level of TIMP1 is indicative of cholangiocarcinoma.

[0021] In a third aspect, the present invention relates to the use of a marker combination comprising TIMP1 and MMP2 in the in vitro assessment of cholangiocarcinoma in a serum, plasma or whole blood sample of an individual, where the levels of TIMP1 and MMP2 are indicative of cholangiocarcinoma. According to the third aspect of the present invention, the levels of TIMP1 and MMP2 may in particular refer to the levels of TIMP1 and MMP2 detected or the detection of TIMP1 and MMP2.

[0022] In a fourth aspect, the present invention provides an in vitro method for assessing cholangiocarcinoma in a patient sample, comprising: (a') determining the level of tissue inhibitor of metalloproteinase 1 (TIMP1) in a patient sample, the patient sample being selected from the group consisting of serum, plasma and whole blood samples from an individual; (b') determining the level of matrix metalloproteinase-2 (MMP2) in patient samples for cholangiocarcinoma; (c') assessing cholangiocarcinoma by comparing the determination results of step (a') and step (b'), where the levels of TIMP1 and MMP2 indicate CCA. According to the fourth aspect of the present invention, the levels of TIPM1 and MMP2 can particularly refer to the levels of TIMP1 and MMP2 detected or the detection of TIMP1 and MMP2.

[0023] In a fifth aspect, the present invention relates to a kit for carrying out at least one of the methods comprising the reagents required for determining the level of TIMP1 determined in step (a) or step (a') and, optionally, for determining the level of MMP2 determined in step (b'). [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 shows the box plot distribution of TIMP1 level values ​​(concentration values ​​in ng / ml) determined from 55 CCA (CCC) samples, 219 HCC samples and 632 risk management subject samples. [Diagram 2] Figure 2 shows the receiver operating characteristic plot (ROC plot, univariate analysis) of CCA vs. risk control samples with AUC of 0.939, and the receiver operating characteristic plot (ROC plot) of CCA vs. HCC samples with AUC of 0.715. X-axis: 1-specificity (false positive); Y-axis: sensitivity (true positive). [Diagram 3] FIG. 3 shows the box plot distribution of TIMP1 level values ​​(concentration values ​​in ng / ml) determined from 55 CCA (CCC) samples and 851 HCC and risk control samples. [Figure 4] Figure 4 shows the receiver operating characteristic plot (ROC plot, univariate analysis) of TIMP1 in differentiating CCA from HCC+ risk control subjects with an AUC of 0.881. X-axis: 1-specificity (false positive); Y-axis: sensitivity (true positive). [Diagram 5] FIG. 5 shows the box plot distribution of multivariate scores of the marker combination TIMP1 and MMP2 from 55 CCA (CCC) and 219 HCC samples. [Figure 6] Figure 6 shows the receiver operating characteristic plot (ROC plot, multivariate analysis) of the marker combination TIMP1 and MMP2 in differentiating CCA from HCC with an AUC of 0.922. X-axis: 1-specificity (false positive); Y-axis: sensitivity (true positive). [Figure 7]FIG. 7 shows the box plot distribution of multivariate scores for the marker combination TIMP1 and MMP2 from 55 CCA (CCC) and 632 risk control samples. [Figure 8] Figure 8: Receiver operating characteristic plot (ROC plot, multivariate analysis) of the marker combination TIMP1 and MMP2 in differentiating CCA from risk control subjects with an AUC of 0.977. X-axis: 1-specificity (false positive); Y-axis: sensitivity (true positive). [Figure 9] FIG. 9 shows box plot distribution of multivariate scores for the marker combination TIMP1 and MMP2 from 55 CCA (CCC) and 851 HCC+ risk control samples. [Figure 10] Figure 10: Receiver operating characteristic plot (ROC plot, multivariate analysis) of the marker combination TIMP1 and MMP2 in differentiating CCA from HCC+ risk control subjects with an AUC of 0.957. X-axis: 1-specificity (false positive); Y-axis: sensitivity (true positive). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description of the Invention Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific embodiments and examples described herein, which may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0026] Several documents are cited herein. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a discrepancy between a definition or teaching of such an incorporated reference and a definition or teaching cited herein, the body of the present specification shall control.

[0027] Each element of the present invention will be described below. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present invention to only the embodiments explicitly described. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application unless the context indicates otherwise.

[0028] definition The word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integers or steps or group of integers or steps.

[0029] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0030] The term "in vitro method" is used to indicate that the method is performed outside an organism, preferably on a body fluid, isolated tissue, organ or cell.

[0031] The term "assessing cholangiocarcinoma (CCA)" is used to indicate that the method according to the invention assists a medical professional, including, for example, a physician, to assess whether an individual has the disease CCA or is at risk of developing the disease CCA.

[0032] A "level of TIMP1 above the reference level" indicates that an individual has the disease CCA or is at risk of developing or prognosing the course of the disease CCA. As used herein, the term "reference level" or reference sample refers to a sample or a level of a sample that is analyzed in a substantially identical manner to the level of a sample or a sample of interest, and the information of which is compared to the information of the sample of interest. The reference level or reference sample thereby provides a standard that allows the evaluation of the information obtained from the sample of interest. The reference level or reference sample may be derived from a healthy or normal tissue, organ, or individual, thereby providing a standard of the health status of the tissue, organ, or individual. The difference between the normal reference sample or normal reference level status and the sample of interest status may indicate the risk of developing a disease or the presence or further progression of such a disease or disorder. The reference sample reference level may be derived from an abnormal or diseased tissue, organ, or individual, thereby providing a standard of the health status of the tissue, organ, or individual. The difference between the abnormal reference sample or abnormal reference level status and the sample of interest status may indicate a reduced risk of developing a disease or the absence or amelioration of such a disease or disorder.

[0033] The term "elevated" or "increased" level of an indicator refers to a higher level of such indicator in a sample compared to the level of such indicator in a reference or reference sample. For example, a protein that is detectable in a higher amount in a fluid sample of an individual afflicted with a given disease than in the same fluid sample of an individual not afflicted with the disease has an elevated level.

[0034] As used herein, the term "biomarker" or "marker" or "biochemical marker" or "marker molecule" refers to a molecule used as a target for analyzing a patient test sample. In one embodiment, an example of such a molecular target is a protein or polypeptide. A protein or polypeptide used as a marker in the present invention is considered to include naturally occurring fragments of the protein, in particular immunologically detectable fragments. An immunologically detectable fragment preferably comprises at least 6, 7, 8, 10, 12, 15 or 20 consecutive amino acids of the marker polypeptide. The skilled artisan will recognize that proteins released by cells or present in the extracellular matrix may be damaged, for example during inflammation, and may be degraded or cleaved into such fragments. Certain markers may be synthesized in an inactive form and subsequently activated by proteolysis. As the skilled artisan will appreciate, a protein or a fragment thereof may also be present as part of a complex. Such a complex may also be used as a marker in the sense of the present invention. Additionally or alternatively, the marker polypeptide may carry a post-translational modification. Examples of post-translational modifications among others are glycosylation, acylation, and / or phosphorylation. As used herein, the term "biomarker" or "marker" generally refers to a molecule, including a gene, a protein, a carbohydrate structure, or a glycolipid, a metabolite, an mRNA, an miRNA, a protein, DNA (cDNA or genomic DNA), DNA copy number, or an epigenetic change, such as an increase, decrease, or change in DNA methylation (e.g., cytosine methylation, or CpG methylation, non-CpG methylation); histone modifications (e.g., (de)acetylation, (de)methylation, (de)phosphorylation, ubiquitination, sumoylation, ADP-ribosylation); a change in nucleosome positioning, whose expression or presence in or on a mammalian tissue or cell can be detected by standard methods (or the methods disclosed herein) and can be predictive, diagnostic, and / or prognostic of the susceptibility of a mammalian cell or tissue to a treatment regimen.

[0035] As used herein, the term "sample" or "patient sample" refers to a biological sample obtained for the purpose of in vitro evaluation. In the methods of the present invention, the sample or patient sample may preferably include any bodily fluid. Test samples include blood, serum, plasma, urine, saliva and synovial fluid. Preferred samples are whole blood, serum or plasma. As the skilled artisan will appreciate, any such assessment is performed in vitro. The patient sample is then discarded. The patient sample is used only for the in vitro methods of the present invention, and no material from the patient sample is returned to the patient's body.

[0036] The term "patient" or "subject" as used herein refers to any single human subject eligible for treatment who is experiencing or has experienced one or more signs, symptoms, or other indicators of the disease CCA. Intended to be included as a subject is any subject involved in a clinical research trial who does not show any clinical signs of the disease, or a subject involved in an epidemiological study, or a subject who has once been used as a control.

[0037] An "individual" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, an individual is a human.

[0038] As used herein, the term "determining" the level of a biomarker refers to quantification of the biomarker, e.g., determining or measuring the level of the biomarker in a sample using appropriate detection methods as described elsewhere herein.

[0039] In certain embodiments, the term "reference level" herein refers to a predefined value. In this context, "level" or "level value" encompasses absolute amounts, relative amounts or concentrations, as well as any value or parameter that can be correlated therewith or derived therefrom. The skilled person will understand that the reference level is predefined and set to meet routine requirements, for example, in terms of specificity and / or sensitivity. These requirements may vary, for example, from one regulatory agency to another. For example, the sensitivity or specificity of an assay may have to be set at a certain limit, for example, 80%, 90%, 95%, or 98%, respectively. These requirements may also be defined in terms of positive predictive value or negative predictive value. Nevertheless, based on the teachings given in the present invention, it is always possible for the skilled person to arrive at a reference level that meets those requirements. In one embodiment, the reference level is determined in a reference sample from a healthy individual. In one embodiment, the reference level is predefined in a reference sample from the disease entity to which the patient belongs. In certain embodiments, the reference level may be set, for example, at any percentage between 25% and 75% of the total distribution of values ​​in the disease to be investigated. In other embodiments, the reference level can be set, for example, to the median, tertile or quartile determined from the entire distribution of values ​​in the reference sample from the disease entity being investigated. In one embodiment, the reference level is set to the median, determined from the entire distribution of values ​​in the disease being investigated. The reference level can vary depending on various physiological parameters, such as age, sex or subpopulation, as well as the means used to determine the biomarkers TIMP1 and, optionally, the combination of TIMP1 and MMP2 referred to herein. In one embodiment, the reference sample is derived from essentially the same type of cell, tissue, organ or body fluid source as the sample from the individual or patient subjected to the method of the invention, for example, when blood is used as a sample for determining the level of the biomarkers TIMP1 and, optionally, MMP2 in an individual according to the invention, the reference level is also determined in blood or a part thereof.

[0040] In certain embodiments, the term "an increased level of a biomarker above or compared to a reference level" refers to a level of a biomarker in a sample from an individual or patient above a reference level or an overall increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or more compared to the reference level as determined by the methods described herein. In certain embodiments, the term increase refers to an increase in the level of a biomarker in a sample from an individual or patient, where the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 75-fold, 80-fold, 90-fold or 100-fold higher compared to a reference level previously determined from a reference sample, for example.

[0041] In certain embodiments, the term "reduction" or "below" herein refers to a level of a biomarker in a sample from an individual or patient below a reference level, or an overall reduction of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more compared to the reference level as determined by the methods described herein. In certain embodiments, the term reduction in the level of a biomarker in a sample from an individual or patient, the reduction in the level is at most about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold or 0.01-fold or less compared to a reference level previously determined from a reference sample, for example.

[0042] The term "indicative of cholangiocarcinoma" or "indicative that an individual has CCA" is used to indicate that the level of the biomarker TIMP1, and optionally the combination of TIMP1 and MMP2, is very informative, but not diagnostic without error. Not all (100%) patients with the disease CCA have a level of the biomarker TIMP1 above the reference level, and not all healthy individuals have a level of the biomarker TIMP1 below the reference level. As the skilled person will understand, in many diseases, biochemical markers do not have 100% specificity and at the same time 100% sensitivity. In such cases, for example, with respect to the level of the biomarker TIMP1 and optionally the combination with MMP2 in the disease CCA, an assessment is made with a certain probability, for example at a given specificity level or at a given sensitivity level. The skilled person will be fully familiar with the mathematical / statistical methods used to calculate specificity, sensitivity, positive predictive value, negative predictive value, reference value or total error. Any of these parameters can be calculated and used to obtain an indication of the presence or absence of the disease CCA.

[0043] As used herein, the term "comparison" refers to comparing the level of a biomarker in a sample from an individual or patient to a reference level of the biomarker as specified elsewhere herein. It should be understood that comparing as used herein generally refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute amount of a reference, whereas a concentration is compared to a concentration of a reference, or an intensity signal obtained from a biomarker in a sample is compared to an intensity signal of the same type obtained from a reference sample. The comparison can be performed manually or computer-assisted. Thus, the comparison can be performed by a computing device (e.g., a system as disclosed herein). The values ​​of the measured or detected levels of the biomarkers in a sample from an individual or patient and the reference level can, for example, be compared to each other, and the comparison can be performed automatically by a computer program that executes an algorithm for the comparison. The computer program that performs the evaluation provides the desired assessment in a suitable output format. For computer-assisted comparison, the determined amount values ​​may be compared by the computer program to values ​​corresponding to suitable standards stored in a database. The computer program may further evaluate the results of the comparison, i.e., automatically provide the desired assessment in a suitable output format. For computer-aided comparison, the determined quantity values ​​may be compared by a computer program with values ​​corresponding to suitable standards stored in a database, which may further evaluate the comparison results, i.e. automatically provide the desired rating in a suitable output format.

[0044] The phrase "comparing the level determined in step (a) to a reference level of TIMP1" is merely used to further illustrate what would be clear to a person skilled in the art anyway. The reference sample can be an internal or external control sample. In one embodiment, an internal reference sample is used, i.e., the marker level(s) are assessed in the test sample as well as one or more other sample(s) taken from the same subject to determine whether there is an alteration in the level(s) of said marker(s). In another embodiment, an external reference sample is used. For an external reference sample, the presence or amount of the marker in a sample from an individual is compared to the presence or amount of the marker in an individual known to suffer from or known to be at risk for a given condition; or in an individual known to be free of a given condition (i.e., "normal individual"). For example, the marker level in a patient sample can be compared to a level known to be associated with a particular course of disease in CCA. Usually, the marker level in the sample is directly or indirectly correlated with a diagnosis, and the marker level is used to determine whether an individual is at risk for CCA, for example. Alternatively, the marker levels of the sample can be compared to marker levels known to be associated with response to treatment, for example, in CCA patients. Depending on the intended diagnostic application, an appropriate reference sample is selected in which a control or reference value of the marker is established. It will be understood by the skilled artisan that such a reference sample in one embodiment is obtained from an age-matched and disease-free reference population. As will be appreciated by the skilled artisan, the absolute marker value established in the reference sample will depend on the assay used. Preferably, samples from 100 well-characterized individuals from an appropriate reference population are used to establish the reference value. A preferred reference population may also be selected to consist of 20, 30, 50, 200, 500 or 1000 individuals. Healthy individuals represent a preferred reference population for establishing a control or reference value.

[0045] As used herein, the phrase "providing an assessment" refers to assessing the CCA of a patient using the generated information or data regarding the level or presence of TIMP1 and optionally MMP2 in a patient's sample. The information or data may be in any form, written, audio, or electronic. In some embodiments, using the generated information or data includes communicating, presenting, reporting, storing, transmitting, transferring, providing, disseminating, administering, or a combination thereof. In some embodiments, the communicating, presenting, reporting, storing, transmitting, transferring, providing, disseminating, administering, or a combination thereof is performed by a computer device, an analyzer unit, or a combination thereof. In some further embodiments, the communicating, presenting, reporting, storing, transmitting, transferring, providing, disseminating, administering, or a combination thereof is performed by a researcher or a medical professional. In some embodiments, the information or data includes an assessment of the level of TIMP1, optionally in combination with MMP2, to an established reference level of TIMP1 and MMP2, respectively. In some embodiments, the information or data includes an indication that TIMP1 and optionally MMP2 are present or absent in the sample. In some embodiments, the information or data includes an indication that the patient was evaluated with CCA.

[0046] The term "antibody and fragments thereof" is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. In particular, antibodies are used herein as "binders". The term "binders" refers to molecules that contain binding moieties that specifically bind to the corresponding target biomarkers TIMP1 and / or MMP2 molecules. Examples of "binders" are nucleic acid probes, nucleic acid primers, DNA molecules, RNA molecules, aptamers, antibodies, antibody fragments, peptides, peptide nucleic acids (PNAs), or chemicals. Thus, antibodies or fragments thereof have specific binding affinity for TIMP1 and MMP2, respectively.

[0047] The terms "specific binding" or "specifically binds" refer to a binding reaction in which the molecules of the binding pair exhibit binding to one another under conditions in which they do not significantly bind to another molecule.

[0048] The terms "specific binding" or "specifically binds" when referring to an antibody or protein or peptide as a binding agent means that the binding agent binds to at least 10 -7 The term "specific binding" or "specifically binding" refers to a binding reaction that binds to a corresponding target molecule with an affinity of at least 10 -8 M, or even more preferably at least 10 -9 The term "specific" or "specifically" refers to the affinity of M. The term "specific" or "specifically" is used to indicate that other molecules present in the sample do not significantly bind to the binding agent specific for the target molecule. Preferably, the level of binding to molecules other than the target molecule results in a binding affinity of no more than 10%, more preferably no more than 5%, of the affinity for the target molecule.

[0049] The term "specific binding" or "specifically binds" when referring to a nucleic acid as a binder refers to a hybridization reaction in which the binder or probe contains a hybridization region that is exactly or substantially complementary to the target sequence of interest. A hybridization assay performed using a binder or probe under sufficiently stringent hybridization conditions allows for selective detection of a specific target sequence. The hybridization region is preferably about 10 to about 35 nucleotides in length, more preferably about 15 to about 35 nucleotides in length. The use of modified bases or base analogs that affect hybridization stability, as is known in the art, may allow for the use of shorter or longer probes with comparable stability. A binder or probe can consist entirely of a hybridization region or can include additional features that allow for detection or immobilization of the probe but do not significantly change the hybridization properties of the hybridization region.

[0050] The terms "specific binding" or "specifically binds", when referring to a nucleic acid aptamer as a binding agent, refer to a binding reaction in which the nucleic acid aptamer binds to a corresponding target molecule with an affinity in the low nM to pM range.

[0051] The term "detectable label" refers to the attachment of a specific tag to an antibody to aid in the detection of the antibody. Numerous labels (also called dyes) are available that can be broadly categorized into the following categories, all of which are grouped together and each of which represents an embodiment according to the present disclosure:

[0052] (a) Fluorescent dye Fluorescent dyes are described, for example, by Briggs et al. "Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058).

[0053] Fluorescent labels or fluorophores include rare earth chelates (europium chelates), fluorescein-type labels (including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein), rhodamine-type labels (including TAMRA), dansyl, lissamine, cyanine, phycoerythrin, Texas Red, and their analogs. Fluorescent labels can be attached to aldehyde groups contained within target molecules using the techniques disclosed herein. Fluorescent dyes and fluorescent labeling reagents include those commercially available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0054] (b) Luminescent dye Luminescent dyes or labels can be further sub-classified into chemiluminescent dyes and electrochemiluminescent dyes.

[0055] Different classes of chemiluminescent labels include systems based on luminol, acridinium compounds, coelenterazine and analogues, dioxetanes, peroxyoxalic acid and peroxyoxalic acid derivatives. For immunodiagnostic procedures, mainly acridinium-based labels are used (a detailed overview is given in Dodeigne C. et al., Talanta 51 (2000) 415-439).

[0056] The main relevant labels used as electrochemiluminescent labels are electrochemiluminescent complexes based on ruthenium and iridium, respectively. Electrochemiluminescence (ECL) has proven to be very useful for analytical applications as a sensitive and selective method. ECL combines the analytical advantages of chemiluminescence analysis (absence of background light signal) with the ease of reaction control by applying an electrode potential. In general, ruthenium complexes, especially [Ru(Bpy)3], which are regenerated with TPA (tripropylamine) in the liquid phase or at the liquid-solid interface, are used. 2+ (emitting photons at about 620 nm) is used as an ECL label. Recently, iridium-based ECL labels have also been described (WO 2012 / 107419 A1).

[0057] (c) The radioactive label is 3 H, 11 C. 14 C. 18 F, 32 P, 35 S, 64 Cu, 68 Gn, 86 Y, 89 Zr, 99 T.C., 111 In, 123 I, 124 I, 125 I, 131 I, 133 Xe, 177 Lu, 211 At or 131 It uses radioactive isotopes (radioactive nuclides) such as Bi.

[0058] (d) Metal chelate complexes suitable as labels for imaging and therapeutic purposes are well known in the art (U.S. Patent Application Publication No. 2010 / 0111856; U.S. Patent No. 5,342,606; U.S. Patent No. 5,428,155; U.S. Patent No. 5,316,757; U.S. Patent No. 5,480,990; U.S. Patent No. 5,462,725; U.S. Patent No. 5,428,139; U.S. Patent No. 5,385,893; U.S. Patent No. 5,739,294; U.S. Patent No. 5,750,660; U.S. Patent No. 5,834,456; Hnatowich et al, J. Immunol. Methods 65 (1983) 147-157; Meares et al, Anal. Biochem. 142 (1984) 68-78; Mirzadeh et al, Bioconjugate Chem.1(1990)59-65;Meares et al,J.Cancer(1990),Suppl.10:21-26;Izard et al,Bioconjugate Chem.3(1992)346-350;Nikula et al,Nucl.Med.Biol.22(1995)387-90;Camera et al. al,Nucl.Med.Biol.20(1993)955-62;Kukis et al,J.Nucl.Med.39(1998)2105-2110;Verel et al.,J.Nucl.Med.44(2003)1663-1670;Camera et al. al,J.Nucl.Med.21(1994)640-646;Ruegg et al,Cancer Res.50(1990)4221-4226;Verel et al,J.Nucl.Med.44(2003)1663-1670;Lee et al,Cancer Res.61(2001)4474-4482;Mitchell,et al,J.Nucl.Med.44(2003)1105-1112;Kobayashi et al Bioconjugate Chem.10(1999)103-111;Miederer et al,J.Nucl.Med.45(2004)129-137;DeNardo et al,Clinical Cancer Research 4(1998)2483-90;Blend et al,Cancer Biotherapy&Radiopharmaceuticals18(2003)355-363;Nikula et al J.Nucl.Med.40(1999)166-76;Kobayashi et al al, J. Nucl. Med. 39 (1998) 829-36; Mardirossian et al, Nucl. Med. Biol. 20 (1993) 65-74; Roselli et al, Cancer Biotherapy & Radiopharmaceuticals, 14 (1999) 209-20). .

[0059] The term "signal from a detectable label of an antibody or a fragment thereof" is used herein to mean an intensity signal obtained from a detectable label of an antibody or a fragment thereof in a sample. The signal can be determined with computer assistance. The signal can be performed by a computing device.

[0060] The term "calculated quantification signal" means here and below that, as standard and routine, TIMP1 and optionally MMP2 are quantified and / or measured via a calibration curve.

[0061] The term "isolated" with reference to an antibody is one that is separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0062] TIMP1 (tissue inhibitor of metalloproteases-1) is a 184 amino acid sialoglycoprotein with a molecular weight of 28.5 kDa (see, for example, Murphy et al Biochem J. 1981, 195, 167-170) that inhibits metalloproteases such as interstitial collagenase MMP1, or stromelysin or gelatinase B. In the understanding of the present invention, the term TIMP-1 encompasses proteins with significant structural homology to human TIMP1, which inhibit the proteolytic activity of metalloproteases. The presence of human TIMP1 can be detected by using antibodies that specifically detect epitopes of TIMP1. TIMP1 can also be determined by detection of related nucleic acids, such as the corresponding mRNA. TIMP1 in the sense of the present invention can be characterized by the sequence shown in SEQ ID NO: 1 or a homologous sequence thereof. In particular, homologous sequences have at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.

[0063] Matrix metalloproteinase-2 (MMP2), also called gelatinase A, is a 66 kDa zinc- and calcium-binding protease that is synthesized as an inactive 72 kD precursor. MMP2 is synthesized by a variety of cells, including vascular smooth muscle cells, mast cells, macrophage-derived foam cells, T lymphocytes, and endothelial cells (Johnson, J Let al., Arterioscler. Thromb. Vasc. Biol. 18:1707-1715, 1998). MMP2 is normally found in plasma complexed with its physiological regulator, TIMP2 (Murawaki, Y. et al., J. Hepatol. 30:1090-1098, 1999). Normal plasma concentrations of MMP2 are approximately less than 550 ng / ml (8 nM). MMP2 expression is elevated in vascular smooth muscle cells within atherosclerotic lesions and can be released into the bloodstream in the case of plaque instability (Kai, H. et al., J. Am. Coll. Gardiol. 32: 368-372. 1998). Furthermore, MMP2 has been implicated as a cause of plaque instability and rupture (Shah, PK et al., Circulation 92: 1565-1569, 1995). Serum MMP2 concentrations are elevated in hospitalized patients with stable angina, unstable angina, and AMI, and the elevation was significantly greater in unstable angina and AMI than in stable angina (Kai, H. et al., J. Am. Coll. Cardiol. 32: 368-372, 1998). There was no change in serum MMP2 concentrations in individuals with stable angina following a treadmill exercise test (Kai, H. et al., J. Am. Coll. Cardiol. 32:368-372, 1998).Serum and plasma MMP2 are elevated in patients with gastric cancer, hepatocellular carcinoma, liver cirrhosis, urothelial carcinoma, rheumatoid arthritis and lung cancer (Murawaki, Y., et al., J. Hepatol. 30:1090-1098, 1999; Endo, K., et al., Anticancer Res. 17:2253-2258, 1997; Gohji, K. et al., Cancer 78:2379-2387, 1996; Gruber, BLet al., Clin. Immunol. Immunopathol. 78:161-171, 1996; Garbisa, S. et al., Cancer Res. 52:4548-4549, 1992). In addition, MMP2 can also be translated from the platelet cytosol into the extracellular space during platelet aggregation (Sawicki, G. et al., Thromb. Haemost. 80:836-839, 1998). MMP2 was elevated in the serum of individuals with unstable angina and AMI at the time of admission, with maximum levels approaching 1.5 μg / ml (25 nM) (Kai, H. et al., J. Am. Coll. Cardiol. 32:368-372, 1998). Serum MMP-2 concentrations peaked 1-3 days after onset in both unstable angina and AMI and began to return to normal after 1 week (Kai, H. et al., J. Am. Coll. Cardiol. 32:368-372, 1998).

[0064] MMP2 in the sense of the present invention can be characterized by the sequence shown in SEQ ID NO: 2 or a homologous sequence thereof. In particular, the homologous sequence has an amino acid sequence identity of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0065] The measured CA19-9 (carbohydrate antigen 19-9) value is defined by the use of the monoclonal antibody 1116-NS-19-9. The 1116-NS-19-9 reactive determinant in serum is expressed mainly on mucin-like proteins that contain multiple CA19-9 epitopes (Magnani JL, Arch. Biochem. Biophys. 426 (2004) 122-131). 3-7% of the population has a Lewis a-negative / b-negative blood group configuration and is unable to express mucin with reactive determinant CA19-9. This must be taken into account when interpreting the findings. CA19-9-containing mucins are expressed in the epithelium of the fetal stomach, intestine and pancreas. It can also be found in low concentrations in adult tissues in the liver, lungs and pancreas (Stieber, P. and Fateh-Moghadam, A., Boeringer Mannheim, Cat. No. 1536869 (engl), 1320947 (dtsch). ISBN 3-926725-07-9 dtsch / engl, Juergen Hartmann Verlag, Marloffstein-Rathsberg (1993); Herlyn, M., et al., J. Clin. Immunol. 2 (1982) 135-140).

[0066] CEA (carcinoembryonic antigen) is a monomeric glycoprotein (molecular weight approximately 180.000 daltons) with a variable carbohydrate content of approximately 45-60% (Gold P. and Freedman SO, J. Exp Med 121 (1965) 439-462). CEA, like AFP, belongs to the group of carcinoembryonic antigens produced during embryonic and fetal life. The CEA gene family consists of about 17 active genes in two subgroups. The first group contains CEA and nonspecific cross-reactive antigens (NCA), and the second group includes pregnancy-specific glycoproteins (PSG). CEA is found mainly in the fetal digestive tract and fetal serum. It also occurs in small amounts in intestinal, pancreatic and hepatic tissues of healthy adults. Since the formation of CEA is suppressed after birth, it is difficult to measure serum CEA levels in healthy adults. High CEA concentrations are frequently found in cases of colorectal adenocarcinoma (Stieber, P. and Fateh-Moghadam, A., supra). Slight to moderate CEA elevations (rarely >10 ng / mL) occur in 20-50% of benign diseases of the intestine, pancreas, liver and lungs (e.g. cirrhosis, chronic hepatitis, pancreatitis, ulcerative colitis, Crohn's disease, emphysema) (Stieber P., and Fateh-Moghadam A., supra). Smokers also have elevated CEA values. The main indication for CEA measurement is the therapeutic management and follow-up of patients with colorectal cancer. Measurement of CEA is not recommended for cancer screening in the general population. CEA concentrations within the normal range do not exclude the possibility of the presence of malignant disease. The antibody in the assay manufactured by Roche Diagnostics reacts with CEA and (as with almost all CEA detection methods) with meconium antigen (NCA2). Cross-reactivity with NCAl is 0.7% (Hammarstrom S., et al., Cancer Res. 49 (1989) 4852-4858; and Bormer OP, Tumor Biol. 12 (1991) 9-15).

[0067] Embodiment In a first aspect, the present invention provides an in vitro method for assessing cholangiocarcinoma in a patient sample, comprising: a) determining the level of tissue inhibitor of metalloproteinase 1 (TIMP1) in a patient sample, the patient sample being selected from the group consisting of serum, plasma and whole blood samples from an individual; b) comparing the level of TIMP1 determined in step (a) with a reference level of TIMP1; and c) assessing cholangiocarcinoma in the patient sample by comparing the level determined in step (a) with a reference level of TIMP1, wherein an increased level of TIMP1 compared to the reference level of TIMP1 is indicative of cholangiocarcinoma in the patient sample.

[0068] The inventors of the present invention have surprisingly been able to demonstrate that marker TIMP1 is useful for assessing CCA.Due to the uncertainty of classifying different stages of CCA, it is quite possible that TIMP1 may become one of the most important criteria in the assessment of patients with CCA in the future.The method of the present invention is suitable for assessing CCA.It has been found that the increase in the level, for example concentration, of TIMP1 in sample compared to normal control indicates CCA.

[0069] In an embodiment of the first aspect of the invention, the method comprises a determining step (a), a comparing step (b) and a CCA assessing step (c). In particular, the method is carried out in the following order: step (a), followed by step (b), followed by step (c).

[0070] In an embodiment of the first aspect of the present invention, the level of tissue inhibitor of metalloproteinase 1 (TIMP1) in a patient sample is determined. The patient sample is selected from the group consisting of serum, plasma and whole blood samples from an individual. In particular, the patient sample is serum. The term "serum" as used herein is the clear liquid portion of blood that can be separated from clotted blood. The term "plasma" as used herein is the clear liquid portion of blood that contains blood cells. Serum differs from plasma, the liquid portion of normal, unclotted blood contains red and white blood cells and platelets. It is the blood clot that makes the difference between serum and plasma. The term "whole blood" as used herein includes all components of blood, such as white and red blood cells, platelets, and plasma. An increase in the level of TIMP1 compared to the baseline level of TIMP1 is indicative of cholangiocarcinoma in the patient sample.

[0071] In an embodiment of the first aspect of the invention, the level of TIMP1 in a patient sample is determined by a sandwich immunoassay (step (a)). Sandwich immunoassays are known to those skilled in the art. In such an assay, a first specific binding agent is used to capture TIMP1 on one side and a second specific binding agent (labeled to be directly or indirectly detectable) is used on the other side. The specific binding agent used in the sandwich assay format can be an antibody specifically directed against TIMP1. On the one hand, detection can be performed by using different capture and label antibodies, i.e. antibodies that recognize different epitopes on TIMP1. On the other hand, a sandwich assay can also be performed with a capture label antibody against the same epitope of TIMP1. In this embodiment, only dimeric and multimeric forms of TIMP1 can be detected. In one embodiment, an antibody against TIMP1 is used in a qualitative (TIMP1 is present or absent) or quantitative (determining the amount of TIMP1) immunoassay. In particular, the level of TIMP1 in a patient sample is determined by an Elecsys assay. The Elecsys assay is known to those of skill in the art and therefore will not be described in detail at this point.

[0072] In an embodiment of the first aspect of the invention, the level of TIMP1 in a patient sample is determined by competitive immunoassay or enzyme-linked immunosorbent assay (ELISA).

[0073] For the determination of TIMP1, the sample obtained from the individual is incubated in vitro with a specific binder for TIMP1 under conditions suitable for the formation of a binder-TIMP1 complex. Such suitable incubation conditions are easily identified by the skilled person without any inventive effort, and there is no need to specify such conditions. The amount of binder-TIMP1 complex is determined and used to assess CCA. As the skilled person will understand, there are many methods for determining the amount of specific binder-TIMP 1 complex, all of which are described in detail in relevant textbooks (see, for example, Tijssen, P. (supra) or Diamandis, EP, and Christopoulos, TK (eds.), Immunoassay, Academic Press, Boston (1996)).

[0074] Immunoassays are well known to those skilled in the art. Methods and practical applications and procedures for carrying out such assays are summarized in relevant textbooks. Examples of relevant textbooks are Tijssen, P., Preparation of enzyme-antibody or other enzyme-macromolecule conjugates, In: Practice and theory of enzyme immunoassays, pp. 221-278, Burdon, RH and v. Knippenberg, PH (eds.), Elsevier, Amsterdam (1990), and various volumes of Colowick, SP, and Caplan, NO, (eds.), Methods in Enzymology, Academic Press (dealing with immunological detection methods, in particular volumes 70, 73, 74, 84, 92 and 121).

[0075] In an embodiment of the first aspect of the invention, the marker TIMP1 is specifically determined in vitro from a liquid sample by use of a specific binding agent.

[0076] In an embodiment of the first aspect of the invention, the specific binding agent is, for example, a receptor for TIMP1, a lectin that binds to TIMP1, an antibody to TIMP1, a peptidomimetic agent to TIMP1, a bispecific double binding agent or a bispecific antibody format. A specific binding agent has at least 10 binding affinities to its corresponding target molecule. 7 A specific binding agent has an affinity of 10 l / mol for its target molecule. 8 It is preferable that the affinity of the ion exchange polymer is 10 9 It is also preferred that the affinity is 1 / mol.

[0077] Those skilled in the art will understand that the term specific is used to indicate that other biomolecules present in the sample do not significantly bind to the binding agent specific for TIMP1 of SEQ ID NO:1. Preferably, the level of binding to biomolecules other than the target molecule results in a binding affinity that is at most no more than 10%, no more than 5%, no more than 2%, or no more than 1% of the affinity for the target molecule, respectively. Preferred specific binding agents meet both of the above minimum criteria for affinity and specificity. Examples of specific binding agents are peptides, peptidomimetics, aptamers, spiegelmers, darpins, ankyrin repeat proteins, Kunitz-type domains, antibodies, single domain antibodies (see Hey T. et al., Trends Biotechnol. 23 (2005) 514-30 522), and monovalent fragments of antibodies.

[0078] For the results as disclosed in the present invention, antibodies from various sources may be used. Standard protocols for obtaining antibodies can also be used, as well as modern alternative methods. Alternative methods for generating antibodies include, among others, the use of synthetic or recombinant peptides that represent clinically relevant epitopes of ASC for immunization. Alternatively, DNA immunization, also known as DNA vaccination, can be used. Obviously, monoclonal or polyclonal antibodies from different species, such as rabbits, sheep, goats, rats or guinea pigs, can be used. Monoclonal antibodies are ideal tools in the development of assays for clinical routine, since they can be produced in any quantity required with certain characteristics.

[0079] In certain preferred embodiments of the first aspect of the invention, the specific binding agent is an antibody or a fragment thereof. The fragment is preferably a monovalent antibody fragment, preferably a monovalent fragment derived from a monoclonal antibody.

[0080] As one of skill in the art will now appreciate, TIMP1 has been identified as a marker, which is useful in assessing CCA. A variety of immunodiagnostic procedures can be used to obtain data comparable to the results of the present invention.

[0081] In an embodiment of the first aspect of the invention, step (a) comprises contacting in vitro a portion of a serum, plasma or whole blood sample from an individual with an antibody or fragment thereof having specific binding affinity for TIMP1, thereby forming a complex between the antibody or fragment thereof and TIMP1 present in the patient sample, the antibody carrying a detectable label; separating the complex formed in the contacting step from the complex-free antibody or fragment thereof; and quantifying a signal from the detectable label of the antibody or fragment thereof comprising the complex formed in the contacting step, the signal being proportional to the amount of TIMP1 present in the individual's patient sample, thereby determining the level of TIMP1 in the individual's sample based on the calculated quantification signal.

[0082] In an embodiment of the first aspect of the invention, step (b) comprises comparing the calculated level value of TIMP1 in the individual patient sample determined in the quantification step with a reference level of TIMP1.

[0083] In an embodiment of the first aspect of the invention, step (c) comprises providing an assessment of cholangiocarcinoma in the individual if the calculated level value of TIMP1 is greater than the reference level of TIMP1.

[0084] In an embodiment of the first aspect of the present invention, steps (a) to (c) described in the last three paragraphs above may be combined with each other.

[0085] In an embodiment of the first aspect of the invention, the antibody or fragment thereof is a non-mammalian antibody. The antibody or fragment thereof is not isolated from a human.

[0086] In an embodiment of the first aspect of the invention, the specific binding agent is preferably an antibody which specifically binds to TIMP1.

[0087] In an embodiment of the first aspect of the invention, the antibody or fragment thereof is isolated from an immunized animal, the animal being selected from the group consisting of mouse, rabbit, sheep, chicken, goat and guinea pig.

[0088] In an embodiment of the first aspect of the invention, TIMP1, in particular the soluble form of TIMP1, is determined in vitro in a suitable sample.Preferably, the sample is derived from a human subject, such as a CCA and / or HHC patient and / or a person at risk of CCA and / or suspected of having CCA and / or a person at risk of HHC and / or suspected of having HHC.

[0089] In an embodiment of the first aspect of the present invention, the sample is obtained from a human subject at risk of being managed. As referred to herein, "a subject at risk of being managed" refers to a liver disease or injury selected from the group consisting of cirrhosis, chronic viral hepatitis, alcoholism, non-alcoholic steatohepatitis, diabetes, obesity, hepatobiliary flukes, primary sclerosing cholangitis (PSC), biliary cysts, hepatolithiasis, toxins, primary biliary cirrhosis (PBC), primary hemochromatosis, and combinations thereof.

[0090] In an embodiment of the first aspect of the invention, the sample is obtained from a human subject at risk of being selected from the group consisting of cirrhosis, chronic viral hepatitis, alcoholism, non-alcoholic steatohepatitis, diabetes, obesity, hepatobiliary flukes, primary sclerosing cholangitis (PSC), biliary cysts, hepatolithiasis, toxins, primary biliary cirrhosis (PBC), primary hemochromatosis, and combinations thereof. In particular, the sample is obtained from a human subject not diagnosed with choledocholithiasis.

[0091] In a first embodiment of the invention, the method is used to differentiate between cholangiocarcinoma and a risk control subject selected from the group consisting of cirrhosis, chronic viral hepatitis, alcohol excess, non-alcoholic steatohepatitis, diabetes, obesity, hepatobiliary flukes, primary sclerosing cholangitis (PSC), biliary cysts, hepatolithiasis, toxins, primary biliary cirrhosis (PBC), primary hemochromatosis, and combinations thereof.

[0092] In an embodiment of the first aspect of the invention, the method is used to differentiate cholangiocarcinoma from hepatocellular carcinoma.

[0093] In an embodiment of the first aspect of the present invention, the value of TIMP1 determined in a control group or population is used to establish the reference range. In a preferred embodiment, the level of TIMP1 is considered to be elevated if the determined value is above the 90% percentile of the reference range. In a further preferred embodiment, the level of TIMP1 is considered to be elevated if the determined value is above the 95% percentile, 96% percentile, 97% percentile or 97.5% percentile of the reference range.

[0094] In an embodiment of the first aspect of the present invention, a sample provided by a patient with already confirmed CCA in a particular situation can be used as a positive control sample, preferably assayed in parallel with the sample to be investigated. In such a setting, a positive result for the marker TIMP1 in the positive control sample indicates that the test procedure has worked at a technical level.

[0095] In an embodiment of the first aspect of the present invention, the patient sample is based on a liquid or body fluid sample obtained from an individual and in vitro determination of TIMP1 in such a sample. As used herein, "individual" refers to a single human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary diseases. Preferably, the individual, control or patient is a human.

[0096] It is known to those skilled in the art that the measurement result of step (a) by the method(s) of the present invention is compared with a reference level of TIMP1. Such a reference level can be determined using a negative reference sample, a positive reference sample, or a mixed reference sample that includes one or more of these types of controls. The negative reference sample preferably includes a sample from a healthy individual or an individual who has not been diagnosed with CCA. The positive reference sample preferably includes a sample from a subject who has a diagnosis of CCA. In an embodiment of the first aspect of the present invention, the positive reference sample or the negative reference sample is obtained from a patient who has not been diagnosed with choledocholithiasis.

[0097] In an embodiment of the first aspect of the invention, the reference sample is a biological sample provided from a reference group of apparently healthy individuals or individuals at risk of developing CCA, in particular for in vitro evaluation. As used herein, the term "reference value" refers to a value established in an apparently healthy individual or a reference group of individuals not affected by or at risk of developing CCA.

[0098] In an embodiment of the first aspect of the present invention, step (c) is performed by a computing device.

[0099] In a second aspect, the present invention relates to the use of TIMP1 as a marker molecule in the in vitro assessment of cholangiocarcinoma in a serum, plasma or whole blood sample of an individual, where a level of TIMP1 above a reference level of TIMP1 is indicative of cholangiocarcinoma. All embodiments described above for the first and / or other aspects of the invention apply to the second aspect of the invention and vice versa.

[0100] In a third aspect, the present invention relates to the use of a marker combination comprising TIMP1 and MMP2 in the in vitro assessment of cholangiocarcinoma in a serum, plasma or whole blood sample of an individual, where the levels of TIMP1 and MMP2 are indicative of cholangiocarcinoma. All embodiments described above for the first and / or second aspect and / or other aspects of the invention apply to the third aspect of the invention and vice versa.

[0101] In an embodiment of the third aspect of the invention, the combination of markers consists of TIMP1 and MMP2, which may mean that there are no other markers to assess CCA.

[0102] In a fourth aspect, the present invention provides an in vitro method for assessing cholangiocarcinoma in a patient sample, comprising: (a') determining the level of tissue inhibitor of metalloproteinase 1 (TIMP1) in a patient sample, the patient sample being selected from the group consisting of serum, plasma and whole blood samples from an individual; (b') determining the level of matrix metalloproteinase-2 (MMP2) in patient samples for cholangiocarcinoma; and (c') assessing cholangiocarcinoma by comparing the determination results of step (a') and step (b'), wherein the levels of TIMP1 and MMP2 are indicative of CCA.

[0103] All embodiments described above for the first, second, third and / or other aspects of the invention apply to the fourth aspect of the invention and vice versa.

[0104] Preferably, the embodiments described for step (a) apply to step (a').

[0105] In an embodiment of the fourth aspect of the present invention, step (c') comprises assessing cholangiocarcinoma by comparing the determination results of step (a') and step (b'), wherein the detected levels of TIMP1 and MMP2 are indicative of CCA.

[0106] In an embodiment of the fourth aspect of the invention, step (a') comprises contacting in vitro a portion of a serum, plasma or whole blood sample from an individual with an antibody or fragment thereof having specific binding affinity for TIMP1, thereby forming a complex between the antibody or fragment thereof and TIMP1 present in the patient sample, the antibody carrying a detectable label; separating the complex formed in the contacting step from the complex-free antibody or fragment thereof; and quantifying a signal from the detectable label of the antibody or fragment thereof comprising the complex formed in the contacting step, the signal being proportional to the amount of TIMP1 present in the individual's patient sample, thereby determining the level of TIMP1 in the individual's sample based on the calculated quantification signal.

[0107] In an embodiment of the fourth aspect of the invention, step (b') comprises contacting in vitro a portion of a serum, plasma or whole blood sample from an individual with an antibody or fragment thereof having specific binding affinity for MMP2, thereby forming a complex between the antibody or fragment thereof and MMP2 present in the patient sample, the antibody carrying a detectable label; separating the complex formed in the contacting step from the complex-free antibody or fragment thereof; and quantifying a signal from the detectable label of the antibody or fragment thereof comprising the complex formed in the contacting step, the signal being proportional to the amount of MMP2 present in the individual's patient sample, thereby determining the level of MMP2 in the individual's sample based on the calculated quantified signal.

[0108] In an embodiment of the fourth aspect of the present invention, steps (a') and (b') as described in the last two paragraphs above may be combined with each other.

[0109] In an embodiment of the fourth aspect of the present invention, step (c') comprises incorporating the level of MMP2 determined in step (b') and the level of TIMP1 determined in step (a') into a statistical methodology to generate an output value indicative of whether the patient sample has cholangiocarcinoma or is at risk of developing cholangiocarcinoma.

[0110] In an embodiment of the fourth aspect of the invention, the statistical methodology used is for example logistic regression.

[0111] In an embodiment of the fourth aspect of the invention, a multivariate score is calculated.

[0112] Other methods may also be used, for example selected from DA (i.e., linear, quadratic, regularized discriminant analysis), kernel methods (i.e., SVM), non-parametric methods (i.e., k-nearest neighbors), PLS (partial least squares), tree methods (i.e., logistic regression, CART, random forests, boosting).

[0113] The performance of the results of the applied statistical methods used according to the present invention can be best described by their receiver operating characteristics (ROC). The ROC curve addresses both the sensitivity (number of true positives) and specificity (number of true negatives) of the test. Thus, the sensitivity and specificity values ​​for a given biomarker or combination of biomarkers are indicators of the performance of the test. For example, if a combination of biomarkers has a sensitivity and specificity value of 80%, then 80 out of 100 diseased patients will be correctly identified from determining the presence of a particular combination of biomarkers as positive for the disease, while 80 out of 100 patients without the disease will be correctly tested negative for the disease.

[0114] A suitable statistical classification model, such as logistic regression, can be derived for the combination of biomarkers with or without clinical variables. Furthermore, the logistic regression equation can be extended to include other (clinical) variables, such as the age and sex of the patient. As before, the ROC curve can be used to access the performance of the logistic regression model in discriminating between patients and controls. The logistic regression equation is a common statistical procedure used in such cases and is preferred in the context of the present invention, although other mathematical or statistical methods, such as decision trees or machine learning procedures, can also be used.

[0115] One convenient goal for quantifying the diagnostic accuracy of a laboratory test is to express its performance in a single number. The most common global measure is the area under the curve (AUC) of the ROC plot. The area under the ROC curve is a measure of the probability that a perceived measurement allows for correct identification of a condition. Values ​​typically range from 1.0 (perfect separation of the two groups of test values) to 0.5 (no obvious distribution difference between the two groups of test values). The area does not depend only on a particular part of the plot, such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. It is a quantitative descriptive expression of how close the ROC plot is to a perfect one (area = 1.0). In the context of the present invention, the two distinct conditions can be whether or not the patient has a cancer such as CCA.

[0116] In an embodiment of the fourth aspect of the present invention, steps (a'), (b') and (c') as set out in the last three paragraphs above may be combined with each other.

[0117] In an embodiment of the fourth aspect of the present invention, each of steps (a') and (b') comprises a sandwich-type immunoassay.

[0118] In an embodiment of the fourth aspect of the present invention, step (c') is performed by a computing device.

[0119] In an embodiment of the fourth aspect of the invention, the sample is obtained from a human subject at risk of being selected from the group consisting of cirrhosis, chronic viral hepatitis, alcoholism, non-alcoholic steatohepatitis, diabetes, obesity, hepatobiliary flukes, primary sclerosing cholangitis (PSC), biliary cysts, hepatolithiasis, toxins, primary biliary cirrhosis (PBC), primary hemochromatosis, and combinations thereof. In particular, the sample is obtained from a human subject not diagnosed with choledocholithiasis.

[0120] In a fourth embodiment of the invention, the method is used to differentiate between cholangiocarcinoma and a risk control subject selected from the group consisting of cirrhosis, chronic viral hepatitis, alcohol excess, non-alcoholic steatohepatitis, diabetes, obesity, hepatobiliary flukes, primary sclerosing cholangitis (PSC), biliary cysts, hepatolithiasis, toxins, primary biliary cirrhosis (PBC), primary hemochromatosis, and combinations thereof.

[0121] In an embodiment of the fourth aspect of the invention the method is used to differentiate cholangiocarcinoma from hepatocellular carcinoma.

[0122] In an embodiment of the fourth aspect of the invention, the specific binding agent is preferably an antibody which specifically binds to MMP2.

[0123] In a fifth aspect, the present invention relates to a kit for carrying out the method according to the first and / or fourth aspect of the invention comprising the reagents required for determining the level of TIMP1 determined in step (a) or (a') and, optionally, for determining the level of MMP2 determined in step (b'). All embodiments described above for the first, second, third, fourth and / or other aspects of the invention apply to the fifth aspect of the invention and vice versa.

[0124] In an embodiment of the fifth aspect of the invention, the kit comprises the reagent(s) necessary to specifically determine the level of TIMP1 and the reagent(s) necessary to determine or measure the marker MMP2 used together in a combination of CCA markers. The kit in one embodiment comprises an antibody or a fragment thereof that specifically binds to TIMP1. In a further embodiment, the antibody fragment in the kit is selected from the group consisting of Fab, Fab', F(ab')2 and Fv. In one embodiment, the invention relates to a kit comprising at least two antibodies or fragments thereof that specifically bind to at least two non-overlapping epitopes contained in the TIMP1 sequence of SEQ ID NO: 1. Preferably, the at least two antibodies or fragments thereof comprised in the kit according to the invention are monoclonal antibodies. The kit in one embodiment further comprises a biochip on which the antibodies or fragments thereof are immobilized.

[0125] In an embodiment of the fifth aspect of the present invention, the reagent for TIMP1 and / or MMP2 is a combination of one or more components, such as probes (e.g., antibodies), controls, buffers, reagents (e.g., conjugates and / or substrates), instructions, etc. disclosed herein.

[0126] A specific exemplary embodiment is as follows.

[0127] 1. An in vitro method for assessing cholangiocarcinoma in a patient sample, comprising: a) determining the level of tissue inhibitor of metalloproteinase 1 (TIMP1) in said patient sample, said patient sample being selected from the group consisting of serum, plasma and whole blood samples from an individual; b) comparing the level of TIMP1 determined in step (a) with a reference level of TIMP1; c) assessing cholangiocarcinoma in the patient sample by comparing the level determined in step (a) to a reference level of TIMP1, wherein an increased level of TIMP1 compared to the reference level of TIMP1 is indicative of cholangiocarcinoma in the patient sample; A method comprising:

[0128] 2. step (a) comprises contacting in vitro a portion of the serum, plasma or whole blood sample from the individual with an antibody or fragment thereof having specific binding affinity for TIMP1, thereby forming a complex between the antibody or fragment thereof and TIMP1 present in the patient sample, the antibody carrying a detectable label; Separating the complex formed in the contacting step from the antibody or fragment thereof that does not contain the complex; and / or quantifying a signal from the detectable label of the antibody or fragment thereof comprising the complex formed in the contacting step, the signal being proportional to the amount of TIMP1 present in the patient sample of the individual, whereby the level of TIMP1 in the sample of the individual is calculated based on the quantified signal; and / or step (b) comprises comparing the calculated level of TIMP1 in the patient sample of the individual determined in the quantifying step with a reference level of TIMP1; and / or 2. The method of embodiment 1, wherein step (c) comprises providing an assessment of cholangiocarcinoma in said individual if the calculated TIMP1 level value is greater than said reference level of TIMP1.

[0129] 3. The method of embodiment 1 or 2, wherein said antibody or fragment thereof is isolated from an immunized animal, said animal being selected from the group consisting of mouse, rabbit, sheep, chicken, goat and guinea pig.

[0130] 4. The method of any one of embodiments 1 to 3, wherein step (a) comprises a sandwich-type immunoassay.

[0131] 5. The method of any one of embodiments 1 to 4, wherein step (c) is performed by a computing device.

[0132] 6. The use of TIMP1 as a marker molecule in the in vitro assessment of cholangiocarcinoma in serum, plasma or whole blood samples of an individual, where a level of TIMP1 above a baseline level of TIMP1 is indicative of cholangiocarcinoma.

[0133] 7. Use of a marker combination comprising TIMP1 and MMP2 in the in vitro assessment of cholangiocarcinoma in a serum, plasma or whole blood sample of an individual, wherein said levels of TIMP1 and MMP2 are indicative of cholangiocarcinoma.

[0134] 8. An in vitro method for assessing cholangiocarcinoma in a patient sample, comprising: (a') determining the level of tissue inhibitor of metalloproteinase 1 (TIMP1) in said patient sample, said patient sample being selected from the group consisting of serum, plasma and whole blood samples from an individual; and (b') determining the level of matrix metalloproteinase-2 (MMP2) for cholangiocarcinoma in said patient sample; (c') assessing cholangiocarcinoma by comparing the results of the determinations of steps (a') and (b'), wherein the levels of TIMP1 and MMP2 are indicative of CCA; and A method comprising:

[0135] 9. step (a') comprises contacting in vitro a portion of the serum, plasma or whole blood sample from the individual with an antibody or fragment thereof having specific binding affinity for TIMP1, thereby forming a complex between the antibody or fragment thereof and TIMP1 present in the patient sample, the antibody carrying a detectable label; Separating the complex formed in the contacting step from the antibody or fragment thereof that does not contain the complex; and / or quantifying a signal from the detectable label of the antibody or fragment thereof comprising the complex formed in the contacting step, the signal being proportional to the amount of TIMP1 present in the patient sample of the individual, whereby the level of TIMP1 in the sample of the individual is calculated based on the quantified signal; and / or step (b') comprises contacting in vitro a portion of the serum, plasma or whole blood sample from the individual with an antibody or fragment thereof having specific binding affinity for MMP2, thereby forming a complex between the antibody or fragment thereof and MMP2 present in the patient sample, the antibody carrying a detectable label; Separating the complex formed in the contacting step from the antibody or fragment thereof that does not contain the complex; 9. The method of embodiment 8, comprising quantifying a signal from the detectable label of the antibody or fragment thereof comprising the complex formed in the contacting step, wherein the signal is proportional to the amount of MMP2 present in the patient sample of the individual, whereby the level of MMP2 in the sample of the individual is calculated based on the quantified signal.

[0136] 10. Step (C') is The method of embodiment 8 or 9, comprising including the level of MMP2 determined in step (b') and the level of TIMP1 determined in step (a') in a statistical methodology to generate an output value indicative of whether the patient sample has or is at risk of developing cholangiocarcinoma.

[0137] 11. The method according to any one of embodiments 8 to 10, wherein each of step (a') and step (b') comprises a sandwich-type immunoassay.

[0138] 12. The method of any one of embodiments 8 to 11, wherein step (c') is performed by a computing device.

[0139] 13. The method according to any one of embodiments 1 to 5 or any one of embodiments 8 to 12 for distinguishing between cholangiocarcinoma and hepatocellular carcinoma.

[0140] 14. The method according to any one of embodiments 1 to 5 or any one of embodiments 8 to 12 for distinguishing between a risk control subject selected from the group consisting of cirrhosis, chronic viral hepatitis, alcohol excess, non-alcoholic steatohepatitis, diabetes, obesity, hepatobiliary flukes, primary sclerosing cholangitis (PSC), biliary cysts, hepatolithiasis, toxins, primary biliary cirrhosis (PBC), primary hemochromatosis, and combinations thereof, and cholangiocarcinoma.

[0141] 15. A kit for carrying out the method according to any one of embodiments 1 to 5 or any one of embodiments 8 to 12, comprising reagents required for determining the level of TIMP1 determined in step (a) or step (a') and, optionally, for determining the level of MMP2 determined in step (b').

[0142] These embodiments are intended to be illustrative and not limiting of the scope of the invention.

[0143] The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit or scope of the invention.

[0144] All references cited herein are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

[0145] Array Description SEQ ID NO: 1 shows the amino acid sequence of TIMP1. (Sequence listing; Uniprot P01033, version 214).

[0146] SEQ ID NO: 2 shows the amino acid sequence of MMP2. (Sequence listing; Uniprot P08253, version 235). EXAMPLES

[0147] The present invention is illustrated merely by the following examples, which should not be construed in any way as limiting the scope of the invention.

[0148] Example 1 Study population: The clinical performance of the biomarkers CEA, CA19-9, TIMP-1 and MMP2 for the diagnosis of cholangiocarcinoma was evaluated in a sample panel consisting of: - 55 CCA samples: 20 intrahepatic, 11 perihilar, 24 no information available. - 219 HCC samples. -632 controls representative of patients at risk for developing CCA and HCC (cirrhosis, chronic viral hepatitis, alcoholic and non-alcoholic steatohepatitis).

[0149] Blood / Serum Collection: Serum samples were collected by the following institutions: Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand; Siriraj Hospital, Bangkok, Thailand; Songklanagarind Hospital, Hat Yai, Songkhla, Thailand; Srinagarind Hospital, Khon Kaen, Thailand; NCT Universitatsklinikum Heidelberg, Heidelberg, Germany, and Vall d'Hebron University Hospital, Barcelona, ​​Spain. This study was conducted in full accordance with the principles of the "Declaration of Helsinki" and with the approval of an independent ethical committee (IEC). Serum samples were collected before procedures (surgery, percutaneous ethanol injection (PEI), chemotherapy, radiotherapy) according to appropriate standard operating procedures (SOPs) and stored at <-70°C until analysis. Repeated freezing and thawing was avoided.

[0150] Serum sample preparation: Serum samples were aspirated into serum tubes and allowed to clot for at least 60 min and up to 120 min at room temperature. After centrifugation (10 min, 2000 g), the supernatant was divided into 1 ml aliquots and frozen at -70°C. Prior to measurement, samples were thawed, re-aliquoted into smaller amounts suitable for the prototype and reference assays, and frozen. Samples were thawed immediately prior to analysis. Thus, each sample within a panel had only two freeze-thaw cycles before measurement.

[0151] Example 2 Assays for the detection of Roche Elecsys® tumor markers: Elecsys® kits were obtained for the tumor markers CEA and CA19-9. All these markers were measured with the Roche Elecsys® in vitro diagnostic. All assays were performed according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany). The concentrations measured by the instrument were used to calculate / generate the AUC data shown in Tables 1 and 5.

[0152] Example 3 ELISA for measuring levels of TIMP1, optionally in combination with levels of MMP2, in human serum or plasma samples: For the measurement of TIMP-1, a commercially available MTP ELISA from R&D Systems Inc. (Minneapolis, USA) was used (Quantikine® ELISA, Human TIMP-1 Immunoassay, Cat. No. DTM100, STM100, PDTM100). Briefly, the assay uses a quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for human TIMP-1 is precoated on a microplate. Standards and samples are pipetted into the wells and any TIMP-1 present is bound by the immobilized antibody. After washing away unbound material, an enzyme-linked polyclonal antibody specific for human TIMP-1 is added to the wells. After washing to remove unbound antibody-enzyme reagent, a substrate solution is added to the wells and color is developed in proportion to the amount of TIMP-1 bound in the first step. The color development is stopped and the color intensity is measured.

[0153] For the measurement of MMP2, a commercially available MTP ELISA from R&D Systems Inc. (Minneapolis, USA) was used (Quantikine® ELISA, Total MMP-2 Immunoassay, Cat. No. MMP200, SMMP200, PMMP200). Briefly, the assay uses a quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for total MMP-2 is precoated onto a microplate. Standards and samples are pipetted into the wells and any MMP-2 present is bound by the immobilized antibody. After washing away unbound material, an enzyme-linked polyclonal antibody specific for MMP-2 is added to the wells. After washing to remove unbound antibody-enzyme reagent, a substrate solution is added to the wells and color is developed in proportion to the amount of MMP-2 bound in the first step. Color development is stopped and the color intensity is measured.

[0154] Example 4 Univariate and multivariate analyses were performed to identify biomarkers / biomarker panels for the best differentiation of: HCC to CCA - From risk management to CCA -HCC+Risk Management to CCA

[0155] Univariate analysis The performance of TIMP1 is evaluated based on its absolute measurement value.The measurement values ​​of all the included patients, both diseased and non-diseased, can be used as threshold value to calculate the sensitivity and specificity at this specific value.Based on the combination of these sensitivity and specificity, ROC curve can be drawn and AUC (area under ROC curve) can be calculated.

[0156] Multivariate analysis The combination of markers TIMP1 and MMP2 is evaluated. Preferably, the values ​​measured for the marker combination TIMP1 and MMP2 are mathematically combined, and the combined value correlates with the underlying diagnostic problem. The marker values ​​can be combined by any suitable state of the art mathematical method.

[0157] Preferably, the method used to correlate the combination of markers of the present invention with, for example, the absence or presence of CCA is a generalized linear model (i.e., logistic regression). However, other methods can also be used, for example selected from DA (i.e., linear, quadratic, regularized discriminant analysis), kernel methods (i.e., SVM), non-parametric methods (i.e., k-nearest neighbors), PLS (partial least squares), tree methods (i.e., logistic regression, CART, random forest, boosting).

[0158] Further details on these statistical methods can be found in the following references: Ruczinski, I., et al, J. of Computational and Graphical Statistics, 12 (2003) 475-511; Friedman, JH, J. of the American Statistical Association 84 (1989) 165-175; Hastie, Trevor, Tibshirani, Robert, Friedman, Jerome, The Elements of Statistical Learning, Springer Series in Statistics (2001); Breiman, L., Friedman, JH, Olshen, RA, Stone, CJ (1984) Classification and regression trees, California: Wadsworth; Breiman, L., Random Forests, Machine Learning, 45 (2001) 5-32; Pepe, MS, The Statistical Evaluation of Medical Tests for Classification and Prediction, Oxford Statistical Science Series 28 (2003); and Duda, RO, Hart, PE, Stork, DG, Pattern Classification, Wiley Interscience, 2nd Edition (2001).

[0159] A preferred embodiment of the present invention is to use cut-offs of an underlying combination of biological markers to distinguish state A from state B, e.g. disease from health. In this type of analysis, the markers are no longer independent but form a marker panel or combination of markers.

[0160] The accuracy of a diagnostic method is best described by its receiver operating characteristics (ROC) (see especially Zweig, MH, and Campbell, G., Clin. Chem. 39 (1993) 561-577). The ROC graph is a plot of all sensitivity / specificity pairs generated by continuously varying the decision threshold over the entire range of observed data.

[0161] The clinical performance of a clinical test depends on its diagnostic accuracy, or ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the ability of a test to correctly distinguish between two different conditions of the subjects studied. Such conditions are, for example, health and disease, or benign versus malignant disease.

[0162] In either case, the ROC plot shows the overlap between the two distributions by plotting sensitivity versus 1-specificity for the entire range of decision thresholds. On the y-axis is the sensitivity, or true positive rate [defined as (number of true positive test results) / (number of true positive + number of false negative test results)]. This is also called positivity in the presence of disease or symptoms. The y-axis is calculated only from the affected subgroup. On the x-axis is the false positive rate, or 1-specificity [defined as (number of false positive results) / (number of true negative + number of false positive results)]. The x-axis is a measure of specificity, calculated only from the unaffected subgroup. The true positive rate and false positive rate are calculated completely separately by using test results from two different subgroups, so the ROC plot is independent of the prevalence of disease in the sample. Each point on the ROC plot represents a sensitivity / 1-specificity pair that corresponds to a particular decision threshold. A test with perfect discrimination (the two distributions of results have no overlap) would have an ROC plot that passes through the upper left corner, with a true positive rate of 1.0, or 100% (perfect sensitivity), and a false positive rate of 0 (perfect specificity). The theoretical plot for a test with no discrimination (the distributions of results for the two groups are identical) would be a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall between these two extremes. (If the ROC plot falls entirely below the 45° diagonal, this is easily corrected by swapping the criterion for "positive rate" from "higher" to "lower" and vice versa.) Qualitatively, the closer the plot is to the upper left corner, the higher the accuracy of the overall test.

[0163] One preferred way to quantify the diagnostic accuracy of a clinical test is to express its performance in a single number. Such an overall parameter is, for example, the so-called "total error" or alternatively the "area under the curve = AUC". The most common global measure is the area under the ROC plot. By convention, this area is always >0.5 (if it is not, the decision rule can be inverted to make it so). Values ​​range from 1.0 (perfect separation of the test values ​​of the two groups) to 0.5 (no obvious distribution difference between the two groups of test values). The area does not depend only on a particular part of the plot, such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the whole plot. It is a quantitative descriptive expression of how close the ROC plot is to the perfect one (area = 1.0).

[0164] A binary logistic regression model is used with log-transformed TIMP1 and MMP2 as independent variables and diagnosis as dependent variable. Log transformation means that both variables are transformed by log10(TIMP1+0.1) and log10(MMP2+0.1) separately. The predictions of the logistic regression model, in this case log odds (probability on a logarithmic scale), are used as multivariate scores. The multivariate scores are treated as continuous covariates from which sensitivity and specificity can be derived for each single threshold.

[0165] Example 5: TIMP1 as a marker for differentiating human CCA vs. HCC, CCA vs. risk control subjects, and CCA vs. risk control + HCC, respectively: Serum concentrations of TIMP1 are evaluated in CCA samples compared to HCC samples and risk control samples representing patients at risk for developing CCA and HCC (cirrhosis, chronic viral hepatitis, alcoholic and non-alcoholic steatohepatitis, 8 cases of biliary cirrhosis and 4 cases of PSC).

[0166] FIG. 1 shows box plots of the distribution of TIMP1 marker level values ​​(TIMP1 concentration values ​​in ng / ml) from 55 CCA (CCC) samples, 219 HCC samples and 632 risk management subject samples.

[0167] Figure 2 shows a receiver operating characteristic plot (ROC plot, single variant analysis) of CCA vs. risk control samples with an AUC of 0.939. Additionally, Figure 2 shows a receiver operating characteristic plot (ROC plot) of CCA vs. HCC samples with an AUC of 0.715.

[0168] FIG. 3 shows a box plot of the distribution of TIMP1 level values ​​(TIMP1 concentration values ​​in ng / ml) determined from 55 CCA (CCC) samples as well as 851 HCC samples including risk control subjects.

[0169] FIG. 4 shows the receiver operating characteristic plot (ROC plot, single variant analysis) of TIMP1 in discriminating CCA from HCC+ risk control subjects with an AUC of 0.881.

[0170] In univariate models, the clinical outcome of TIMP1 is significantly better with respect to the reference markers CA19-9 and CEA, respectively. The levels or concentrations of TIMP1 in serum or plasma of CCA patients are significantly increased compared to HCC patients. A better discrimination of the concentrations of TIMP1 is observed between CCA patients and the control group consisting of patients at risk of developing CCA and HCC (liver cirrhosis, chronic viral hepatitis, alcoholic and non-alcoholic steatohepatitis).

[0171] The clinical performance of TIMP-1 for the diagnosis of CCA exceeds that of the reference markers CA19-9 and CEA (see Table 1): a) For differential diagnosis of CCA vs HCC: TIMP-1 AUC 0.715, CA19-9 AUC 0.672, CEA AUC 0.615. b) For CCA versus risk-managed diagnoses including cirrhosis, chronic viral hepatitis, alcoholic and nonalcoholic steatohepatitis: TIMP-1 AUC 0.939, CA19-9 AUC 0.784, CEA AUC 0.485. c) For diagnosis of CCA vs. HCC and risk management: TIMP-1 AUC 0.881, CA 19-9 AUC 0.755, CEA AUC 0.518. [Table 1]

[0172] We surprisingly found that this novel blood biomarker, TIMP1, is highly sensitive and specific for the non-invasive diagnosis of CCA. Metalloprotease inhibitor 1 TIMP-1 (Uniprot P 01033) has not been previously evaluated in association with cholangiocarcinoma.

[0173] Example 6 Marker combinations: TIMP1 and MMP2 as marker combinations for differentiating human CCA vs. HCC, CCA vs. risk control subjects, and CCA vs. risk control + HCC, respectively: In multivariate analysis, the combination of TIMP-1 and MMP2 was selected as the best model, outperforming all other marker combinations, based on the AUC of each combination of the two biomarkers included in the analysis.

[0174] FIG. 5 shows box plots of the distribution of multivariate scores based on TIMP1 and MMP2 from 55 CCA (CCC) and 219 HCC samples.

[0175] FIG. 6 shows the receiver operating characteristic plot (ROC plot, multivariate analysis) of the marker combination TIMP1 and MMP2 in differentiating CCA from HCC with an AUC of 0.922.

[0176] FIG. 7 shows box plots of the distribution of multivariate scores based on TIMP1 and MMP2 from 55 CCA (CCC) and 632 risk-managed samples.

[0177] FIG. 8 shows the receiver operating characteristic plot (ROC plot, multivariate analysis) of the marker combination TIMP1 and MMP2 in distinguishing CCA from risk management subjects with an AUC of 0.977.

[0178] FIG. 9 shows box plots of the distribution of multivariate scores for the marker combination TIMP1 and MMP2 from 55 CCA (CCC) and 851 risk control samples.

[0179] FIG. 10 shows the receiver operating characteristic plot (ROC plot, multivariate analysis) of the marker combination TIMP1 and MMP2 in differentiating CCA vs. HCC+ risk control subjects with an AUC of 0.957.

[0180] Clinical performance of the combination of TIMP-1 and MMP2 markers for the diagnosis of CCA: a) For differential diagnosis of CCA vs HCC: AUC 0.922. b) For CCA versus risk-managed diagnoses including cirrhosis, chronic viral hepatitis, alcoholic and non-alcoholic steatohepatitis: AUC 0.977. c) For the diagnosis of CCA vs. HCC and risk management: AUC 0.957.

[0181] The determined AUC values ​​are summarized in Table 5. [Table 2]

Claims

1. 1. An in vitro method for aiding in assessing cholangiocarcinoma in a patient sample, comprising: a) determining the level of tissue inhibitor of metalloproteinase 1 (TIMP1) in said patient sample, said patient sample being selected from the group consisting of serum, plasma and whole blood samples from an individual; b) comparing the level of TIMP1 determined in step (a) with a reference level of TIMP1; c) providing a result of the comparison in step (b) to aid in assessing cholangiocarcinoma in said patient sample by comparing said level determined in step (a) with said reference level of TIMP1, wherein an elevated level of TIMP1 compared to said reference level of TIMP1 is indicative of cholangiocarcinoma in said patient sample; A method comprising:

2. step (a) comprises contacting in vitro a portion of the serum, plasma or whole blood sample from the individual with an antibody or fragment thereof having specific binding affinity for TIMP1, thereby forming a complex between the antibody or fragment thereof and TIMP1 present in the patient sample, the antibody carrying a detectable label; Separating the complex formed in the contacting step from the antibody or fragment thereof that does not contain the complex; and / or quantifying a signal from the detectable label of the antibody or fragment thereof comprising the complex formed in the contacting step, wherein the signal is proportional to the amount of TIMP1 present in the patient sample of the individual, whereby the level of TIMP1 in the sample of the individual is calculated based on the quantified signal; and / or step (b) comprises comparing the calculated level of TIMP1 in the patient sample of the individual determined in the quantifying step with a reference level of TIMP1; and / or The method of claim 1, wherein step (c) comprises providing information to assist in assessing cholangiocarcinoma in the individual if the calculated TIMP1 level value is greater than the reference level of TIMP1.

3. 3. The method of claim 2, wherein the antibody or fragment thereof is isolated from an immunized animal, the animal being selected from the group consisting of mouse, rabbit, sheep, chicken, goat and guinea pig.

4. The method of claim 1 , wherein step (a) comprises a sandwich-type immunoassay.

5. The method of claim 1 , wherein step (c) is performed by a computing device.

6. 1. An in vitro method for aiding in assessing cholangiocarcinoma in a patient sample, comprising: (a') determining the level of tissue inhibitor of metalloproteinase 1 (TIMP1) in said patient sample, said patient sample being selected from the group consisting of serum, plasma and whole blood samples from an individual; (b') determining the level of matrix metalloproteinase-2 (MMP2) for cholangiocarcinoma in said patient sample; (c') comparing the determination results of step (a') and step (b') to provide a comparison result for assisting in assessing cholangiocarcinoma, the comparison result being obtained by comparing the level of TIMP1 determined in step (a') with a reference level of TIMP1 and comparing the level of MMP2 determined in step (b') with a reference level of MMP2, providing a comparison result for assisting in assessing cholangiocarcinoma. A method comprising:

7. An in vitro method for aiding in assessing cholangiocarcinoma in a patient sample, comprising: (a') determining the level of tissue inhibitor of metalloproteinase 1 (TIMP1) in said patient sample, said patient sample being selected from the group consisting of serum, plasma and whole blood samples from an individual; (b') determining the level of matrix metalloproteinase-2 (MMP2) for cholangiocarcinoma in said patient sample; (c') comparing the determination results of step (a') and step (b') and providing a comparison result to aid in assessing cholangiocarcinoma, comprising including the level of MMP2 determined in step (b') and the level of TIMP1 determined in step (a') in a statistical methodology to generate an output value indicative of whether the patient sample has or is at risk of developing cholangiocarcinoma; A method comprising:

8. step (a') comprises contacting in vitro a portion of the serum, plasma or whole blood sample from the individual with an antibody or fragment thereof having specific binding affinity for TIMP1, thereby forming a complex between the antibody or fragment thereof and TIMP1 present in the patient sample, the antibody carrying a detectable label; Separating the complex formed in the contacting step from the antibody or fragment thereof that does not contain the complex; and / or quantifying a signal from the detectable label of the antibody or fragment thereof comprising the complex formed in the contacting step, wherein the signal is proportional to the amount of TIMP1 present in the patient sample of the individual, whereby the level of TIMP1 in the sample of the individual is calculated based on the quantified signal; and / or step (b') comprises contacting in vitro a portion of the serum, plasma or whole blood sample from the individual with an antibody or fragment thereof having specific binding affinity for MMP2, thereby forming a complex between the antibody or fragment thereof and MMP2 present in the patient sample, the antibody carrying a detectable label; Separating the complex formed in the contacting step from the antibody or fragment thereof that does not contain the complex; The method of claim 6 or 7, comprising quantifying a signal from the detectable label of the antibody or fragment thereof comprising the complex formed in the contacting step, wherein the signal is proportional to the amount of MMP2 present in the patient sample of the individual, whereby the level of MMP2 in the sample of the individual is calculated based on the quantified signal.

9. The method according to claim 6 or 7, wherein each of the steps (a') and (b') comprises a sandwich-type immunoassay.

10. The method of claim 6 or 7, wherein step (c') is performed by a computing device.

11. 10. The method of claim 1 as a method for aiding in distinguishing between cholangiocarcinoma and hepatocellular carcinoma.

12. 13. The method of claim 1 as a method for assisting in differentiating between cholangiocarcinoma and a risk management subject selected from the group consisting of cirrhosis, chronic viral hepatitis, alcohol excess, nonalcoholic steatohepatitis, diabetes, obesity, hepatobiliary flukes, primary sclerosing cholangitis (PSC), biliary cysts, hepatolithiasis, toxins, primary biliary cirrhosis (PBC), primary hemochromatosis, and combinations thereof.

13. A kit for carrying out the method according to any one of claims 1 to 5 or any one of claims 7 to 12, comprising the reagents required for determining the level of TIMP1 determined in step (a) or step (a').

14. The kit of claim 13, comprising reagents required for determining the level of MMP2 determined in step (b').

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